1. Ssaha_pileup - a SNP/indel detection pipeline from new sequencing data
Zemin Ning
Sequence Assembly & Analysis S 1

2. Outline of the Talk: Newly released ssaha2 version 2.1
Alignment presentation - cigar format
Process of single and pair end reads
Transcriptome reads
Ssaha_pileup consensus
Solexa homopolymers
Indel dection
Future work 2

3. Alignment Presentation – “cigar” Format
CIGAR format (Stabenau et al 2004) is a compact representation of the patterns of gapped alignments. It adds more complexity in order to parse the alignment in the later stage using relative less space.
./ssaha2 -output cigar subject query.fastq
query_name subject_name M 57 D 1 M 36 I 2 M 26

4. Pipeline of ssaha_pileup
Indel File
Pipeline of ssaha_pileup
Sequencing
Reads
Reference
fasta
Ssaha_indel
SNP File
Pileup/cons
Alignment - ssaha2 SE
Ssaha_cigar
ssaha_pileup PE
Ssaha_pairs
Ssaha_clean
Unique placed
cigar read file

5. Mapping Score in ssaha2
Read mapping score is used to assess the repetitive feature of the read in the genome. In the cigar file cigar::50 Smap = 50 is the mapping score:
R = read length; Smax - maximum alignment score (smith-waterman) of the hits on genome; Smax2 - second best alignment score of the hits on genome; Say you have one read of 30 bases which has a few hits on the genome: Best hit: exact match with Smax 30; Second best hit: one base mismatch with Smax2 29. The mapping score for this read is Smap = 10;
Read
Reference 29 21 30 25 14 27

6. SNP Confidence Score in ssaha2
SNP score is calculated as the sum of weighted read mapping scores, combined with base quality.
For Solexa reads:
Smap - read mapping score, from 0 (repeat) to 50 (unique);
Fq - base quality factor: Fq = 1 if Q>=30 Fq = 0.5 if Q<30;
N – number of read coverage at the location. 17

7. Transcriptome Reads with “-trans 1”
Short Reads
Intron
Reference Sequence

8. C.elegans Transcriptome Assembly
Solexa reads:
Number of reads: 3,565,445; Transcriptome size: ,933,960 bp;
Read length: 35;
Estimated read coverage: 3.1X; Number of covered bases: 15,774,889 bp;
Number of single coverage: 5,422,688.
Assembly features:
Number of contigs:
Total assembled bases: Mb
N50 contig size:
Largest contig:
Averaged contig size:
Contig coverage: %
Contig extension errors: 0
Mis-assembly errors: 0

9. Read Mapping vs De novo Assembly
Chromosome I:
Chromosome V:

10. Pileup/cons File Format

11. SNP output File Format

12. Indels or Homopolymers ?

13. Indels or Homopolymers ?

14. Indels or Homopolymers ?

15. Homopolymers at High Depth

16. P.Faciparum 3D7 Simulations
Indel Detection
P.Faciparum 3D7 Simulations
Simulated Solexa reads:
Number of reads: ,647,985 Genome size: Mbp
Read length: 36
Read coverage: 40x
Num. of uniquely placed PE reads: 24,303,362 Percentage of placed PE reads: 94.5%
Num. of uniquely placed SE reads: 23,229,651
Percentage of placed SE reads: 90.6%
Detection results:
Number of deletions: ,816
Number of detected deletions: 5,668 (97.5%)
Number of false positives: (2.3%)
Number of insertions: ,816
Number of detected insertions: 5,458 (93.8%)
Number of false positives: 15 (0.26%)

17. SNP/indel Detection Human Chromosome X
Simulated Solexa reads:
Number of reads: m Chromosome size: Mbp
Read length: bp
Read coverage: 45x
Number of uniquely placed reads: m
Percentage of placed reads: 75.0%
Detection results:
Total Hom Hez
Number of SNPs: 97,895 30,286 67,609
Percentage in dbSNP: 83.7% 91.7% 80.1%
Number of deletions: 5,597
Number of insertions: 4,294
1_Base 2_base 3_base
Deletion length:
Insertion length:

18. Availability More information:
ftp://ftp.sanger.ac.uk/pub/zn1/ssaha_pileup/
More information:
ftp://ftp.sanger.ac.uk/pub/zn1/ssaha_pileup/ssaha_pileup-readme

19. Acknowledgements: Yong Gu Ben Blackburne Hannes Ponstingl Tony Cox
Quan Long and Other ssaha users